Time evolution of PEG-shedding and serum protein coronation determines the cell uptake kinetics and delivery of lipid nanoparticle formulated mRNA

Development of efficient lipid nanoparticle (LNP) vectors remains a major challenge towards broad clinical translation of RNA therapeutics. New lipids will be required, but also better understanding LNP interactions with the biological environment. Herein, we model protein corona formation on PEG-ylated DLin-MC3-DMA LNPs and identify time-dependent maturation steps that critically unlock their cellular uptake and mRNA delivery. Uptake requires active serum proteins and precedes after a significant (~2 hours) lag-time, which we show can be eliminated by pre-incubating LNPs for 3-4 hours in serum-containing media. This indicates an important role of protein corona maturation for the pharmacokinetic effects of these LNPs. We show, using single-nanoparticle imaging, NMR diffusometry, SANS, and proteomics, that the LNPs, upon serum exposure, undergo rapid PEG-shedding (~30 minutes), followed by a slower rearrangement of the adsorbed protein layer. The PEG-shedding coincides in time with high surface abundance of Apolipoprotein A-II, whereas the LNPs preferentially bind Apolipoprotein E when their maximum uptake-competent state is reached. Finally, we show that pre-incubation of the LNPs enables rapid uptake and allows pulse-chase video-microscopy colocalization experiments with sufficiently short pulse durations to gain improved mechanistic understanding of how intracellular trafficking events determine delivery efficacy, emphasizing early endosomes as important delivery-mediating compartments.

Magnetic Particle Imaging and its Application

Magnetic particle imaging was introduced in 2005 as a new tomographic medical imaging modality and is still under development. Magnetic particle imaging determines the spatial distribution of magnetic nanoparticles by their interaction with an external excitation magnetic field. Therefore, there is no ionizing radiation dose in this trace-based modality. Magnetic nanoparticle imaging provides characteristics, including high spatial and temporal resolution, high sensitivity, expected from an ideal imaging method, and it is also an inherently quantitative method. In this paper, the properties of magnetic fields and particles used in Magnetic particle imaging, as well as its applications are discussed.

Visualizing Electron Beam-Capping Ligand Reactions for Controlled Nanoparticle Imaging with Liquid Phase Transmission Electron Microscopy

Liquid phase transmission electron microscopy (LP-TEM) enables real-time imaging of nanoparticle self-assembly, formation, and etching with single nanometer resolution. Despite the importance of organic nanoparticle capping ligands in these processes, the effect of electron beam irradiation on surface bound and soluble capping ligands during LP-TEM imaging has not been investigated. Here we use correlative LP-TEM and fluorescence microscopy (FM) to demonstrate that polymeric nanoparticle ligands undergo competing crosslinking and chain scission reactions that non-monotonically modify ligand coverage over time. Branched polyethylenimine (BPEI) coated silver nanoparticles were imaged with dose-controlled LP-TEM followed by labeling their primary amine groups with fluorophores to visualize the local thickness of adsorbed capping ligands. FM images showed that free ligands crosslinked in the LP-TEM image area over imaging times of tens of seconds, enhancing local capping ligand coverage on nanoparticles and silicon nitride membranes. Nanoparticle surface ligands underwent chain scission over irradiation times of minutes to tens of minutes, which depleted surface ligands from the nanoparticle and silicon nitride surface. Conversely, solutions of only soluble capping ligand underwent successive crosslinking reactions with no chain scission, suggesting nanoparticles enhanced the chain scission reactions by acting as radiolysis hotspots. The addition of a hydroxyl radical scavenger, tert-butanol, eliminated chain scission reactions and slowed the progression of crosslinking reactions. These experiments have important implications for performing controlled and reproducible LP-TEM nanoparticle imaging as they demonstrate the electron beam can significantly alter ligand coverage on nanoparticles in a non-intuitive manner. They emphasize the need to understand and control the electron beam radiation chemistry of a given sample to avoid significant perturbations to the nanoparticle capping ligand chemistry, which are invisible in electron micrographs.<br>

Visualizing Electron Beam-Capping Ligand Reactions for Controlled Nanoparticle Imaging with Liquid Phase Transmission Electron Microscopy

Liquid phase transmission electron microscopy (LP-TEM) enables real-time imaging of nanoparticle self-assembly, formation, and etching with single nanometer resolution. Despite the importance of organic nanoparticle capping ligands in these processes, the effect of electron beam irradiation on surface bound and soluble capping ligands during LP-TEM imaging has not been investigated. Here we use correlative LP-TEM and fluorescence microscopy (FM) to demonstrate that polymeric nanoparticle ligands undergo competing crosslinking and chain scission reactions that non-monotonically modify ligand coverage over time. Branched polyethylenimine (BPEI) coated silver nanoparticles were imaged with dose-controlled LP-TEM followed by labeling their primary amine groups with fluorophores to visualize the local thickness of adsorbed capping ligands. FM images showed that free ligands crosslinked in the LP-TEM image area over imaging times of tens of seconds, enhancing local capping ligand coverage on nanoparticles and silicon nitride membranes. Nanoparticle surface ligands underwent chain scission over irradiation times of minutes to tens of minutes, which depleted surface ligands from the nanoparticle and silicon nitride surface. Conversely, solutions of only soluble capping ligand underwent successive crosslinking reactions with no chain scission, suggesting nanoparticles enhanced the chain scission reactions by acting as radiolysis hotspots. The addition of a hydroxyl radical scavenger, tert-butanol, eliminated chain scission reactions and slowed the progression of crosslinking reactions. These experiments have important implications for performing controlled and reproducible LP-TEM nanoparticle imaging as they demonstrate the electron beam can significantly alter ligand coverage on nanoparticles in a non-intuitive manner. They emphasize the need to understand and control the electron beam radiation chemistry of a given sample to avoid significant perturbations to the nanoparticle capping ligand chemistry, which are invisible in electron micrographs.<br>

High-efficiency and water-quenching-resistant Tb3+-based nanoparticles for single-particle imaging

The structure of the host lattice has a substantial influence on the optical properties of lanthanide-doped luminescent materials. Hexagonal-phase (β-phase) NaREF4 (RE = rare earth) is the most commonly used crystal structure for lanthanide-doped upconversion nanoparticles (UCNPs) owing to its high upconversion (UC) efficiency. In this work, we report, for the first time, that more efficient cooperative sensitization upconversion (CSU) can be achieved in cubic-phase (α-phase) NaREF4 UCNPs instead of their β-phase counterparts. With the passivation of an inert shell, the UC emission intensity of α-NaYbF4:Tb40%@CaF2 is 10.5 times higher than that of β-NaYbF4:Tb40%@NaYF4. We propose that the high-symmetry crystal structure of the α phase facilitates the formations of [Yb–Yb] dimers and [Yb–Yb–Tb] clusters, which are particularly beneficial for CSU. Moreover, we prove that such Tb3+-based UCNPs are almost impervious to water quenching because of the large energy gap (∼15,000 cm−1) that existed in Tb3+ between its lowest emit-ting level (5D4) and next low-lying level (7F0). Finally, their potential application for single-nanoparticle imaging has also been demonstrated. As expected, the α-core-shell UCNPs measured at the single-nanoparticle level are estimated to be 9-fold brighter than their β-core-shell counterparts. Importantly, the α-NaYbF4:Tb40%@CaF2 UCNPs offer exciting opportunities for realizing single-nanoparticle imaging at ultralow irradiance (30 W/cm2).

The Spectroscopic Properties and Microscopic Imaging of Thulium-Doped Upconversion Nanoparticles Excited at Different NIR-II light

Lanthanide-doped upconversion nanoparticles (UCNPs) are promising bioimaging nanoprobes due to their excellent photostability. As one of the most commonly-used lanthanide activators, Tm3+ ions have perfect ladder-type electron configuration and can be directly excited by bio-friendly near-infrared-II (NIR-II) wavelengths. Here, the emission characteristics of Tm3+-doped nanoparticles under laser excitations of different near-infrared-II wavelengths were systematically investigated. The 1064 nm, 1150 nm and 1208 nm lasers are proposed to be three excitation strategies with different response spectra of Tm3+ ions. Particularly we found that 1150 nm laser excitation enables intense three-photon 475 nm emission, which is nearly 100 times stronger than that excited by 1064 nm excitation. We further optimized the luminescence brightness after investigating the luminescence quenching mechanism of bare NaYF4:Tm (1.75%) core. After growing inert shell, ten-fold increase of emission intensity was achieved. Combining the advantages of NIR-II wavelength and the higher-order nonlinear excitation, a promising facile excitation strategy was developed for the application of thulium-doped upconversion nanoparticles in single nanoparticle imaging and cancer cell microscopic imaging.